The Mechanism Research Of Zheng Gan Fang Formula Relying On AFP Against Precancerous Lesion Of Liver

Objective:RNAi technology was used to construct the model cell line（AFPsiRNA-HepG₂cell line）which is out of expression of the AFP of HepG₂cellline; To investigate The effects of exogenous alpha fetoprotein onproliferation and apoptosis in AFPsiRNA-HepG₂cells;and furtherexplore the specific mechanisms of Zheng Gan Fang formula oninhibiting precancerous lesion of liver.Methods: This paper includes four parts.1.The drug sera was collected from rats. Preliminary screening incytotoxicity and density of the drug sera was determined using thealamar blue method. HepG₂cells were cultured with drug sera, Cellproliferation was assayed by MTT method,GGT was detected by Kitassay,Chemiluminiscence method and Western blot assay were appliedto detect the expression of AFP. Nuclear morphometry wasinvestigated by IFA, Cell cycle and apoptosis was assayed by flowcytometry（FCM）, Real Time PCR were applied to detect the expressionof Cell telomerase gene. 2.Gene sequence target AFP was designed from the website that ishttps://rnaidesigner.invitrogen.com/rnaiexpress/.The loopstructure in the template used TTCAAGAGA to avoid a terminationsigna.Positive Sense strand:5’-T-（GN18）-（TTCAAGAGA）-（N18C）-TTTTTTC-3’,Antisense strand:3’-A（CN18）-（AAGTTCTCT）-（N18G）-AAAAAAGAGCT-5’.Choose four targetspots,named AFP-639，AFP-1020，AFP-1432，AFP-1811. Synthesis oftarget sequence, Build and identifying plasmid, packaging andpurifing viral vector, Transfection, Cell count Computingefficiency of infection, Use Western blot and Real Time PCR methodto detect the gene and protein expression of AFP in all groups.Select interfere with the highest efficiency group, Large amountsof virus package build AFPsiRNA-HepG₂cell lines. And the expressionof. the gene and protein expression of AFP in AFPsiRNA-HepG₂celland after the wedding was detected by Western blot and Real TimePCR method.3.AFPsiRNA-HepG₂cell lines were cultured with different levelsof exogenous AFP.Cell proliferation was assayed by MTT method, GGTwas detected by Kit assay; The nucleus morphological observationwas investigated by fluorescent assay, Cell cycle and apoptosis wasassayed by flow cytometry（FCM）, Real Time PCR were applied to detectthe expression of cell telomerase.4.AFPsiRNA-HepG₂cells was Divided into eight groups:5%of normallybig rat sera,5%of drug sera,10%of normally big rat sera,10%of drugsera,AFP+5%of normally big rat sera,AFP+5%of drug sera, AFP+10%ofnormally big rat sera,AFP+10%of drug sera, cultured24hours, Cellproliferation was assayed by MTT method,GGT was detected by Kitassay,Chemiluminiscence method and Western blot assay were applied to detect the expression of AFP.Nuclear morphometry wasinvestigated by IFA,cell cycle and apoptosis was assayed by flowcytometry（FCM）,Real Time PCR were applied to detect the expressionof cell telomerase gene.Results:1.The results showed that concentrations of20%with normally bigrat sera and drug sera have obvious cytotoxic effect,so theconcentration of5%and10%were selected,and clture for24hours；Compared with the group of normally big rat sera，the concentrationof5%and10%groups have obvious effect in inhibiting cellproliferation, blocking the cell cycle, and inducing cell apoptosis,down-regulating the expression of AFP gene and protein,anddown-regulating the expression of telomerase gene;And have obviouseffect on changeing the shape and structure of the nucleus; Drugsera have no influence on the expression of GGT.2.Four target sequences were choesed, Adenovirus transfectionafter a small amount of packaging,Computing efficiency ofinfection,detecting the gene and protein expression of AFP,theresults suggest that the target spot of “AFP-639”have the highestinterference efficiency（97%） compare withWild type humanhepatocellular carcinoma HepG₂cells.So the the target spot of“AFP-639” was selected to build cell model of AFPsiRNA-HepG₂，the model cells after the wedding can still steady silence theexpression of AFP gene and protein.3.High dose AFP（≥100ug/ml） can inhibit AFPsiRNA-HepG₂cellproliferation, block the cell cycle, cut the telomerase geneexpression and induce cell death, low dose AFP（＜100ug/ml） canpromote cell proliferation, shorten the cell cycle.Different dose AFP have no influence on the expression of GGT.4.Compared with the group of normally normal sera，the concentrationof5%and10%drug sera have obvious effect to inhibit cellproliferation, block the cell cycle in G₀/G₁, Speriod, prolong thecell cycle, significantly induced cell death, significantly cut thetelomerase gene expression, the concentration of5%and10%drugsera groups have no significantly influence on the expression ofGGT.Conclusion:1.Zheng Gan Fang Formula has obvious effect on reducing theexpression of AFP,inhibiting cell proliferation, blocking the cellcycle, and inducing cell apoptosis of HepG₂cells in vitro.2.RNAi technology can be used to build HepG₂cell line out ofexpression of AFP.3.The low concentration of AFP has effect on promoting cellproliferation of AFPsiRNA-HepG₂cells.4.Zheng Gan Fang Formula prevent and treat precancerous lesion ofliver throug restraining the effect on promoting cell proliferitionof AFP.